WO2021013040A1 - 基于微孔阵列芯片的数字pcr扩增装置和利用其进行扩增的方法 - Google Patents

基于微孔阵列芯片的数字pcr扩增装置和利用其进行扩增的方法 Download PDF

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WO2021013040A1
WO2021013040A1 PCT/CN2020/102374 CN2020102374W WO2021013040A1 WO 2021013040 A1 WO2021013040 A1 WO 2021013040A1 CN 2020102374 W CN2020102374 W CN 2020102374W WO 2021013040 A1 WO2021013040 A1 WO 2021013040A1
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microwell
array chip
pcr amplification
microwell array
chip
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PCT/CN2020/102374
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English (en)
French (fr)
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张志峰
王彦文
王晓飞
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成都万众壹芯生物科技有限公司
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Publication of WO2021013040A1 publication Critical patent/WO2021013040A1/zh

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/52—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips
    • B01L9/527—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips for microfluidic devices, e.g. used for lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • C12M1/38—Temperature-responsive control
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the invention belongs to the field of nucleic acid detection, and particularly relates to a digital PCR detection method based on a micropore array chip.
  • Digital PCR can use large-scale parallel PCR amplification to extract weak amplification signals from background noise, and count the number of amplified molecules by "presence or absence of end-point signal”. So far, a variety of dPCR devices have been developed, mainly including well plate type and droplet type.
  • the well-plate dPCR is to discretize nucleic acid molecules on a plate with a sufficient number of microwell arrays of the same size to realize digital amplification.
  • the QuantStudioTM 3D dPCR system launched by ThermoFisher adopts this method. It etches up to 20,000 hexagonal micro-holes on a square plate with a side length of 10mm, each with a volume of 0.8nL, which realizes isolation between the micro-holes. By simply painting and loading, each micro-hole has about 1 target Nucleic acid molecules: After PCR, the fluorescence signal in the well can be detected, and the number of target molecules can be calculated by Poisson distribution.
  • the droplet dPCR uses tens of thousands of monodisperse droplets to divide the sample.
  • the droplets use hydrophobic pipes and oil phase liquids to disperse DNA with each water-in-oil droplet.
  • one droplet It contains only one DNA molecule; after DNA amplification, it can be transferred to a fluorescence detection device for final detection.
  • the disadvantage of this method is that the droplets are likely to merge with each other during the PCR process or the transfer process, resulting in inaccurate results.
  • an object of the present invention is to provide a digital PCR amplification device based on a microwell array chip and a method for amplification using the same.
  • the digital PCR amplification device has the advantages of high detection sensitivity, high accuracy and low cost.
  • the present invention provides a digital PCR amplification device based on a microwell array chip.
  • the digital PCR amplification device includes: a microwell array chip.
  • the microwell array chip includes:
  • a sealing cover the sealing cover being placed above the bottom plate for sealing each microwell in the microwell array
  • a plurality of biochemical sensors one biochemical sensor is arranged in each microwell in the microwell array, and a capture probe is arranged on the surface of the biochemical sensor, and the capture probe is suitable for capturing the in the microwell Target nucleic acid and generate electrical signals;
  • An electrode the electrode being adapted to provide a voltage to the solution in the micropore.
  • the biochemical sensor of the digital PCR amplification device of the above embodiment of the present invention captures the amplified target nucleic acid molecule through the capture probe provided on its surface, and replaces it with the electronic detection method that detects the inherent negative charge of the nucleic acid molecule.
  • the above-mentioned device of the present application can significantly reduce the complexity, volume and price of the system.
  • the present invention can greatly reduce the volume of micropores (which can be reduced to 1 fL to 10 pL), thereby significantly reducing the minimum reagent usage and the price of consumables.
  • the digital PCR amplification device based on the microwell array chip according to the above embodiment of the present invention may also have the following additional technical features:
  • the electrode includes at least one of an on-chip electrode, a solution electrode, and an external electrode, wherein the on-chip electrode or the solution electrode is disposed on the bottom plate.
  • the biochemical sensor is one of ion-sensitive field effect transistors, carbon nanotubes, silicon nanowires, graphene or molybdenum disulfide transistor sensors, or a miniature electrochemical sensor.
  • the surface of the biochemical sensor is provided with a passivation layer, the passivation layer is formed on the inner wall of the micropore, and the capture probe is arranged on the surface of the passivation layer .
  • the passivation layer is a stack of one or more of gold, aluminum oxide, hafnium dioxide, titanium dioxide, tantalum pentoxide, silicon dioxide, and silicon carbide.
  • 1 to 1 million capture probes are provided on the surface of each biochemical sensor.
  • a reading device which is electrically connected to a computer, so as to realize the electrical signal of the biochemical sensor in the microwell before and after PCR amplification of the microwell array chip Read.
  • the reading device includes:
  • a base, a groove for accommodating the microwell array chip is formed on the upper surface of the base, and a metal probe is arranged in the bottom wall of the groove, and the metal probe is suitable for interacting with the microwell array
  • the pin pad of the chip forms an electrical connection, so as to realize the power supply to the micropore array chip and the data reading of the electrical signal of the biochemical sensor;
  • a circuit board the circuit board is arranged on the lower surface of the base, the circuit board includes a data acquisition module, an ADC chip, and a processor connected in sequence, wherein,
  • the data collection module is connected to the metal probe and is suitable for collecting electrical signals of the biochemical sensor
  • the electrical signal is delivered to the ADC chip, and the ADC chip performs analog-to-digital conversion;
  • the electrical signal after the analog-to-digital conversion is sent to the processor, and the processor performs digital signal processing and calculates the concentration of amplified DNA;
  • the upper cover one side edge of the upper cover is hinged to the edge of the base, and is used to cover the base.
  • the reading device includes:
  • a plug-in circuit board includes a storage compartment for accommodating a micro-hole array chip, a cover placed at the mouth of the storage compartment, and a signal for amplifying the biochemical sensor signal on the micro-hole array chip Amplifier chip and plug;
  • a data processing circuit board the data processing circuit board includes a socket, an ADC analog-to-digital conversion chip, a microprocessor MCU, and a communication port.
  • the socket is matched with the plug and is connected to the socket through the plug.
  • the ADC analog-to-digital conversion chip is connected to the signal amplifier chip and the microprocessor MCU, and the microprocessor MCU is connected to the The terminal equipment is connected.
  • it further includes:
  • a thermal cycler the thermal cycler includes an end cover and a base, the end cover is movably mounted on the top of the base;
  • the base includes a placement cavity for accommodating the microwell array chip, close to A heat conduction block provided at the bottom of the placement cavity, a temperature sensor placed at the upper end of the heat conduction block close to the placement cavity, a heater placed at the bottom of the heat conduction block, and a heater placed at the bottom of the heater Radiator;
  • the two sides of the heat conduction block are fixed in the base by a limit plate, the heater is fixed at the bottom of the heat conduction block by a positioning plate, the temperature sensor and the heater are connected with each other through a control circuit
  • the main control board is connected.
  • the present invention also proposes a method for amplification using the digital PCR amplification device of the previous embodiment.
  • the method includes:
  • the present invention also proposes a method for performing amplification using the digital PCR amplification device of the previous embodiment.
  • the method includes:
  • microwell array chip after the PCR amplification reaction is cleaned, it is placed in a reading device, and the standard solution is added to the microwell, and then the target electrical signal of the microwell array chip is read;
  • step (5) is performed according to the following steps:
  • the capture probe of the biochemical sensor does not capture the target nucleic acid, and the difference between the biochemical sensor in the microwell before and after the PCR amplification reaction
  • the value of the electrical signal is 0, and the wells that have not undergone PCR amplification are negative reaction units;
  • the capture probe of the biochemical sensor captures the target nucleic acid, and the biochemical sensor in the micropore before and after the PCR amplification reaction
  • the value of the difference electrical signal is the first electrical signal, and the microwell where a single amplification reaction of the DNA sample occurs is a positive reaction unit;
  • the PCR amplification reaction occurs for a plurality of the DNA samples in the micropore;
  • step (5-2) is performed according to the following steps:
  • step (5-1) After the DNA sample with the concentration to be tested is subjected to the PCR amplification reaction, first calculate the number of all the positive reaction units on the microwell array chip according to step (5-1), and then draw Calculate the initial concentration of the DNA sample from the standard curve.
  • step (5-2) is performed according to the following steps:
  • the number of positive reaction units or the number of negative reaction units obtained by the histogram analysis is taken into the Poisson equation to calculate the initial concentration of the DNA sample.
  • FIG. 1 is a schematic diagram of the body structure of a microwell array chip according to an embodiment of the present invention
  • Fig. 2 (a) a schematic structural diagram of a chip holder type reading device according to an embodiment of the present invention (a perspective view in a closed state);
  • FIG. 2 (b) a schematic structural diagram of a chip holder type reading device according to an embodiment of the present invention (a top view of a closed state);
  • Fig. 2 (c) a schematic structural diagram of a chip holder type reading device according to an embodiment of the present invention (closed state front view);
  • Fig. 2 (d) a schematic structural diagram of a chip holder type reading device according to an embodiment of the present invention (side view in an open state);
  • Fig. 2 (e) a schematic structural diagram of a chip holder type reading device according to an embodiment of the present invention (a perspective view in an open state);
  • FIG. 2 is a cross-sectional view taken along the line A-A in (c) in FIG. 2;
  • FIG. 3 is a block diagram of the composition of a chip holder type reading device according to an embodiment of the present invention.
  • Fig. 4 (a) is a schematic diagram of the overall structure of the card reader according to the embodiment of the present invention.
  • FIG. 4 (b) is a schematic diagram of the structure of the card circuit board in the card reader device of the embodiment of the present invention.
  • Fig. 5 is a block diagram of the composition of the card reader according to the embodiment of the present invention.
  • Figure 6 is a schematic structural diagram of a thermal cycler according to an embodiment of the invention.
  • Fig. 7 is a three-dimensional schematic diagram of a thermal cycler according to an embodiment of the present invention.
  • FIG. 8 is a schematic diagram of method (1) of semiconductor chip micropore PCR in an embodiment of the present invention.
  • FIG. 9 is a schematic diagram of method (2) of semiconductor chip micropore PCR in an embodiment of the present invention.
  • Fig. 10 (a) a schematic diagram of the PCR machine adapter of the embodiment 1 of the present invention.
  • FIG. 10(b) is a schematic cross-sectional view of the PCR machine adapter of FIG. 10(a);
  • FIG. 10(c) is a partial enlarged schematic diagram of the PCR machine adapter in FIG. 10(b);
  • Fig. 11 is a digital two-dimensional thermodynamic diagram of exemplary data of embodiment 1 of the present invention.
  • Figure 12 The electrical signal changes (I/V curve) before and after DNA probe connection (ie, before and after surface chemistry) of Example 1 of the present invention
  • Example 13 is a histogram of sensor signal changes before and after PCR amplification in Example 1 of the present invention.
  • Fig. 14 is a graph of electrical signal changes (I/V curve) of positive units before and after PCR in Example 1 of the present invention.
  • Figure 15 is a standard curve diagram of the number of positive units and the DNA concentration in Example 1 of the present invention.
  • the present invention provides a digital PCR amplification device based on a microwell array chip.
  • the digital PCR amplification device includes: a microwell array chip, and the microwell array chip includes:
  • a biochemical sensor one biochemical sensor is arranged in each microwell 112 in the microwell array 111, and a capture probe 131 is arranged on the surface of the biochemical sensor, and the capture probe is suitable for capturing the microwell
  • the biochemical sensor of the digital PCR amplification device of the above-mentioned embodiment of the present invention captures the amplified target nucleic acid molecules by providing capture probes on its surface, and replaces fluorescence with the electronic detection method that detects the inherent negative charge of the nucleic acid molecules.
  • the above-mentioned device of the present application can significantly reduce the complexity, volume and price of the system.
  • the present invention can greatly reduce the volume of micropores (which can be reduced to 1 fL to 10 pL), thereby significantly reducing the minimum reagent usage and the price of consumables.
  • the electrode includes at least one of an on-chip electrode 141, a solution electrode 142 and an external electrode 143, wherein the on-chip electrode 141 or the solution electrode 142 is disposed on the bottom plate 110.
  • the on-chip electrode 141 or the solution electrode 142 is disposed on the bottom plate 110.
  • the microwell array 111 contains 10,000 to 10 million microwells 112, and the volume of each microwell 112 is 1fl-10pL.
  • the sealing cover 120 is used for compression and sealing, so that the liquids in different micropores 112 are isolated from each other, and there is one target nucleic acid molecule in each micropore 112, thereby realizing single-molecule PCR amplification of DNA samples.
  • the biochemical sensor may be one of an ion sensitive field effect transistor (ISFET), nanowire, graphene, or molybdenum disulfide transistor sensor, or a miniature electrochemical sensor.
  • ISFET ion sensitive field effect transistor
  • biochemical sensors sample a new type of nanowire as the channel field effect transistor nanowire FET or graphene or molybdenum disulfide and other two-dimensional semiconductor materials as the channel FET.
  • these nanotransistor sensors can provide higher Sensitivity, thereby providing a smaller reaction chamber, higher integration and accuracy; miniature electrochemical sensors can greatly reduce the production cost of the amplification device.
  • an ion sensitive field effect transistor includes a metal floating gate structure 132, a channel 133, a source electrode 134, a drain electrode 135 and a silicon substrate 136.
  • ISFET ion sensitive field effect transistor
  • the surface of the biochemical sensor is provided with a passivation layer 150 formed on the inner wall of the microhole 112, and the capture probe 131 is connected to the surface of the passivation layer on.
  • the surface area of the biochemical sensor you can set at least one capture probe and a maximum of 1 million capture probes.
  • nanowires can be coated with one to dozens of capture probes; if it is a relatively large surface area, such as a micropore area 2 A micrometer can be coated with thousands of capture probes.
  • the method of connecting the capture probe 131 to the surface of the passivation layer can adopt a surface chemistry method to connect the capture probe to the surface of the passivation layer.
  • the sulfhydryl DNA probe is directly connected to the surface of the passivation layer.
  • biotin biotin
  • avidin avidin
  • Capture probes can also be formed firmly on the surface of the passivation layer; in other embodiments, an amine group is attached to one end of the DNA, and then the amine group is combined with a single molecule coated with an aldehyde group on the microporous array chip , So as to be fixed to the surface of the passivation layer.
  • the passivation layer can not only prevent the solution in the micropores from contacting other structures such as the biochemical sensor, ensure the accuracy of the test, and prevent the metal material surface in the biochemical sensor from being corroded by the solution; moreover, the passivation layer can also be used for bearing Capture probe.
  • the passivation layer 150 is a stack of one or more of gold, aluminum oxide, hafnium oxide, titanium dioxide, tantalum pentoxide, silicon dioxide, and silicon carbide. Therefore, the passivation layer does not react with the solution.
  • the digital PCR amplification device of the above embodiment of the present invention further includes: a reading device, which is electrically connected to a computer, so as to realize the biochemical sensor in the microwell before and after the PCR amplification of the microwell array chip Reading of electrical signals.
  • the reading device may be a chip holder-type reading device 200 based on a chip holder, and the chip holder-type reading device 200 specifically includes: The base 210, the circuit board 220 and the upper cover 230.
  • the circuit board 220 is arranged on the lower surface of the base 210, and one side edge of the upper cover is hinged to the edge of the base to cover the base.
  • a groove 211 for accommodating the microwell array chip 100 is formed on the upper surface of the base 210, and a metal probe 212 is provided in the bottom wall of the groove 211, and the metal probe 212 is suitable for contacting with
  • the pin pads of the micropore array chip are electrically connected, so as to realize the power supply to the micropore array chip 100 and the data reading of the electrical signals of the biochemical sensor 130;
  • the circuit board 220 includes a data acquisition module, an ADC chip, and a processor (not shown) connected in sequence, wherein the data acquisition module is connected to the metal probe and is suitable for collecting the biochemical
  • the electrical signal of the sensor is transmitted to the ADC chip, and the ADC chip performs analog-to-digital conversion; the electrical signal after the analog-to-digital conversion is transmitted to the processor, and the processor Perform digital signal processing and calculate the concentration of amplified DNA.
  • the processor of the circuit board 220 may be FPGA, DSP, or ARM.
  • the reader may be a card reader based on a card chip PCB circuit board
  • the device 300 specifically includes a card circuit board 310 and a data processing circuit board 320.
  • the card plug-in circuit board 310 includes a container 311 for accommodating a microwell array chip, a cover 312 placed at the mouth of the container, and a signal for amplifying the biochemical sensor 130 on the microwell array chip 100.
  • the pins of the biochemical sensor 130 are soldered on the card circuit board 310 to realize the circuit connection of the biochemical sensor 130.
  • the signal amplifier chip 313 amplifies the electrical signal output by the biochemical sensor 130 to improve the signal-to-noise ratio.
  • the ADC analog-to-digital conversion chip 322 transmitted to the data processing circuit board 320;
  • the data processing circuit board 320 includes a socket 321, an ADC analog-to-digital conversion chip 322, a microprocessor MCU323, and a communication port.
  • the socket 321 is matched with the plug 314, and is connected to the socket through the plug 314.
  • the connection of 321 realizes the communication between the plug-in card circuit board 310 and the data processing circuit board 320.
  • the ADC analog-to-digital conversion chip 322 is connected to the signal amplifier chip 313 and the microprocessor MCU323 respectively, and the microprocessor MCU323 passes through the communication port.
  • the communication port may include the USB port 324, the serial port 325, or the parallel port 326 shown in (b) of FIG. 4.
  • the terminal device can be a computer or a smart phone.
  • the digital PCR amplification device of the foregoing embodiment of the present invention may further include a thermal cycler 400.
  • the microwell array chip 100 can be placed on the thermal cycler 400 to perform the amplification reaction.
  • the thermal cycler 400 includes an end cover 410 and a base, the end cover 410 is movably mounted on the top of the base;
  • the base includes a placement cavity 420 for accommodating the microwell array chip body 100, a tight A heat conduction block 430 disposed at the bottom of the placement cavity 420, a temperature sensor 440 disposed at the upper end of the heat conduction block 430 at a position close to the placement cavity 420, a heater 450 placed at the bottom of the heat conduction block 430, and
  • the temperature sensor 440 and the heater 450 are connected to the main control board 490 through a control circuit.
  • the main control board 490 sets the temperature in the placement cavity 420 according to the needs of the PCR amplification reaction.
  • the temperature sensor 440 is used to monitor the temperature in the placement cavity 420 and transmit it to the main control board 490.
  • the actual temperature in 420 sends a heating or stopping command to the heater 450; the heater 450 may be a semiconductor heater or a thermal resistance.
  • the present invention also proposes a method for amplification using the digital PCR amplification device of the previous embodiment.
  • the method (1) includes:
  • the above step (1) adding samples specifically includes: diluting or concentrating the target DNA fragment 3 according to the concentration in its original sample to a certain ratio, and then adding 5 pairs of primers and an amplification enzyme 4
  • the buffer solution is mixed uniformly and then added to the micropores 112 on the micropore array chip, and is tightly sealed with the sealing cover 120.
  • Each micropore 112 forms a mutually isolated PCR reaction unit, as shown in FIG. 8.
  • Step (3) amplification specifically includes: Step (1)
  • the microwell array chip 100 after sample loading can perform constant temperature PCR and variable temperature PCR amplification reactions, such as placing the microwell array chip 100 in a variable temperature PCR machine or a thermal cycler Variable temperature PCR amplification reaction can be realized on the 400, and the microwell array chip 100 can be placed on a constant temperature PCR machine or a constant temperature table to realize a constant temperature PCR amplification reaction; the sealed microwell array chip 100 can be placed on a PCR machine or a thermal cycler
  • the PCR amplification reaction is carried out in the container; the target DNA fragment 3 in each microwell 112 is subjected to the chain PCR amplification reaction under the action of the primer pair 5 and the amplification enzyme 4, the copy number continues to increase, and is continuously captured and probed.
  • the needle recognizes the capture and releases the electrical signal at the same time.
  • the PCR machine is a flat-plate PCR or a PCR adapter is used for amplification in a traditional PCR machine, such as a 96-well plate.
  • Step (4) The signal reading specifically includes: placing the body of the microwell array chip after PCR amplification reaction in the groove of the reading device, reading the electrical signal of the biochemical sensor at the bottom of the microwell, and obtaining the expansion after data processing.
  • the increased DNA concentration is displayed on the computer or display screen.
  • the upper cover is first opened, the body of the microwell array chip in the sealed state after the PCR amplification reaction is put into the groove on the base, and the upper cover is covered so that the sealed cover compresses the microwells; the microwell array chip
  • the pin pad of the biochemical sensor at the bottom of the main body is electrically connected with the probe in the base, and then the probe is connected to the circuit board to supply power to the main body of the microwell array chip; then the data acquisition module collects the electric power of the biochemical sensor at the bottom of the microwell
  • the signal is converted by the ADC chip, it is sent to FPGA, DSP, ARM and other processors for digital signal calculation processing, and then the calculated initial concentration of the DNA sample is sent to the display screen for display.
  • step (5) is performed according to the following steps:
  • step (5-2) is performed according to the following steps:
  • step (5-1) After the DNA sample with the concentration to be tested is subjected to PCR amplification reaction, first calculate the number of all positive reaction units on the microwell array chip according to step (5-1), and then calculate the DNA sample's value according to the pre-drawn standard curve The initial concentration.
  • step (5-2) can also be performed according to the following steps:
  • the number of positive reaction units or the number of negative reaction units obtained from the histogram analysis is taken into the Poisson equation to calculate the initial concentration of the DNA sample.
  • the present invention also proposes a method for performing amplification using the digital PCR amplification device of the previous embodiment.
  • the method (2) includes:
  • step (5) in the method (2) is the same as the operation analysis step in the step (5) in the method (1) described above, and will not be repeated here.
  • the microporous array chip body with CMOS ISFET as a biochemical sensor is used as an example for specific description.
  • the specific indicators are shown in Table 1:
  • Detection of PCR signal type The hydrogen ion concentration, or the charge of the amplified DNA
  • the operation of connecting the capture probe (also called DNA probe) to the microwell array chip is as follows: a. Mix the connection solution containing the capture probe (see Table 2) and add 2.5uL dropwise to the chip B. Put the microwell array chip into a vacuum device to vacuum (0.1M Pa) and keep it for 1 minute before taking it out; c. After keeping it at room temperature for 1 hour, wash off the unconnected capture probe with deionized water.
  • microwell array chip 100 (also referred to as the chip) into the PCR cone adapter shown in Figure 10; d.
  • the sealing cover seals the surface of the semiconductor chip and closes the PCR adapter upper cover 1030, and presses and seals the sealing sheet with the upper surface of the micropores of the micropore array chip 100, so that each micropore of the micropore array chip 100 is an independent reaction space.
  • PCR tapered adapters can be individually or multiplely placed in the heating tank of the PCR instrument for heating. Liquid for PCR amplification; PCR tapered adapter has good thermal conductivity, which can quickly conduct heat from the heating tank of the PCR instrument to the chip; the chip can be detachably set on the PCR tapered adapter, so that the chip PCR adapter can complete different chips PCR amplification.
  • the PCR cone adapter includes: a base 1010, an upper cover 1030, a plug 1040, a buckle 1060, and a spring 1070. Specifically: an upper cover 1030 buckled and connected to the base 1010 is provided on the upper cover 1030.
  • the plug 1040 on the chip and the outer side of the upper cover 1030 are provided with a buckle 1060 that is buckled on the base.
  • the middle of the buckle 1060 is pivotally connected to the upper cover 1030, the upper end of the buckle 1060 is fixedly connected to one end of the spring 1070, and the other end of the spring 1070 is fixed to the upper cover 1030.
  • the adapter base is made of metal or thermal conductive material to conduct heat conduction on the chip body for PCR amplification.
  • the amplification conditions are shown in Table 4 Shown:
  • Method a Take out the chip and place it in In the socket of the reading device shown in Figures 2 and 3, the circuit of the reading device (provided the reference voltage for the micropore solution of the chip through the on-chip electrode) is directly connected to the chip pins, and the electrical signals of all the sensors in the micropores After data processing, it is read out to the computer or to the display screen; in order to prevent the change of the PCR solution after the PCR amplification in method a, two signals may occur, one is the charge signal of the DNA itself, and the other is the PCR solution itself Another false signal caused by the change, which leads to inaccurate measurement results.
  • each time point 260,000 data are generated, as a frame, corresponding to 26 biochemical sensors.
  • the data is 512 rows ⁇ 512 columns to form a two-dimensional heat map, as shown in Figure 11, the color of each pixel corresponds to the size of the output signal of the corresponding sensor, that is, the pH value/DNA charge in the corresponding micropore.
  • Multiple time points can generate multiple frames of two-dimensional heat maps.
  • (1) Before and after DNA probe connection First, obtain several frames of two-dimensional heat map data of the chip before and after DNA probe connection, and compare the multiple frames of electrical signals before and after DNA probe connection in the microwell. As shown in Fig. 12, the electrical signal of the microwell changes significantly, that is, the DNA probe is connected in the microwell.
  • the upper curve A1 in Fig. 12 indicates before the DNA probe is connected, and the lower curve A2 indicates after the DNA probe is connected.
  • the number of positive sensors calculated based on the signal of the sample to be tested on the chip body is brought into the standard curve equation to calculate the initial copy concentration (C2) of the DNA sample.
  • the measured DNA copy concentration C2 is closer to the actual copy concentration C1, thus verifying that the obtained standard curve is consistent with the Poisson distribution formula.

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Abstract

本发明公开了一种基于微孔阵列芯片的数字PCR扩增装置和利用其进行扩增的方法，其中该数字PCR扩增装置包括：微孔阵列芯片，该微孔阵列芯片包括：底板、密封盖、多个生化传感器和电极，该底板上形成有微孔阵列；该密封盖置于该底板上方，用于密封该微孔阵列中的每个微孔；该微孔阵列中每个微孔底部均设置有一个该生化传感器，该生化传感器上设置有捕获探针，该捕获探针适于捕获所在微孔内的目标核酸并产生电信号；该电极适于向该微孔内的溶液提供电压。该数字PCR扩增装置具有检测灵敏度高、准确度高和成本低的优点。该扩增的方法包括：(1)将DNA反应体系滴加到该微孔阵列芯片中的每个微孔内，并用该密封盖密封；(2)将加样后的该微孔阵列芯片放入读写装置中，读出该微孔阵列芯片的初始电信号；(3)将加样后的该微孔阵列芯片置于热循环器上进行PCR扩增反应；(4)将该PCR扩增反应后的微孔阵列芯片置于读取装置内，利用该读取装置读取该微孔底部生化传感器的目标电信号；(5)通过数据处理后获得DNA样本的初始浓度，并显示在终端设备上。

Description

基于微孔阵列芯片的数字PCR扩增装置和利用其进行扩增的方法 技术领域
本发明属于核酸检测领域，尤其涉及一种基于微孔阵列芯片的数字PCR检测方法。
背景技术
现代生物学研究，特别是医学研究时常涉及对核酸定量分析的需求。比如检测血液中某些游离DNA及其含量，可以指导某些癌症的临床诊断，以及监控癌症的治疗效果。以往的定量主要采取荧光定量PCR进行相对定量，即选取一个表达稳定的基因的转录物作为参照，来评判目的核酸的量的高低。但这种方法易受非目的核酸分子(背景噪音)的干扰，只适用于定性或者低精度检测，无法满足某些含量特别稀少的目标核酸分子的检测。
数字PCR(dPCR)则能通过大规模的平行PCR扩增，将微弱的扩增信号从背景噪音中提炼出来，以“终点信号的有或无”来统计被扩增的分子的个数。到目前为止，已经发展出了各式各样的dPCR装置，主要有孔板式和液滴式。
孔板式dPCR就是在足够数量的同尺寸微孔阵列排布的平板上离散化核酸分子，实现数字化扩增。例如：ThermoFisher公司推出的QuantStudioTM 3D dPCR系统就是采用这种方式。它在一块10mm边长正方形平板上蚀刻出高达20000个六角形微孔，每个微孔容积0.8nL，实现微孔间隔离，通过简单涂刷上样，每个微孔有1个左右的目标核酸分子；PCR后就可检测孔中荧光信号，通过泊松分布计算目标分子的个数。
液滴式dPCR则是通过数以万计的单分散性小液滴来分割样本，液滴借助疏水的管道和油相液体，把DNA分散与各个油包水小液滴，一个液滴理论上只含有一个左右的DNA分子；DNA扩增后可以转移到荧光检测装置进行最终检测。该方法的缺点是液滴容易在PCR过程或者转移过程中出现液滴相互汇合的情况，导致结果不准确。
以上各种dPCR的技术都采用荧光信号来读取结果，这就要求配套装置中含有昂贵的荧光激发装置和耗材、荧光高分辨率检测装置。另外，光信号的相互污染也会增加判断结果难度。
因此，目前现有的PCR扩增装置仍有待进一步改进。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本发明的一个目的在于提出一种基于微孔阵列芯片的数字PCR扩增装置和利用其进行扩增的方法。该数字PCR扩增装置具有检测灵敏度高、准确度高和成本低的优点。
为此，根据本发明的一个方面，本发明提出了一种基于微孔阵列芯片的数字PCR扩增装置，根据本发明的实施例，该数字PCR扩增装置包括：微孔阵列芯片，所述微孔阵列芯片包括：
底板，所述底板上形成有微孔阵列；
密封盖，所述密封盖置于所述底板上方，用于密封所述微孔阵列中的每个微孔；
多个生化传感器，所述微孔阵列中每个微孔内均设置有一个所述生化传感器，所述生化传感器表面上设置有捕获探针，所述捕获探针适于捕获所在微孔内的目标核酸并产生电信号；
电极，所述电极适于向所述微孔内的溶液提供电压。
由此，本发明上述实施例的数字PCR扩增装置的生化传感器通过其表面设置的捕获探针来捕获扩增后的目标核酸分子，通过检测核酸分子的固有负电荷的电子检测方式，来代替荧光检测的传统方式。本申请的上述装置可以显著降低系统的复杂度、体积和价格。另外，本发明可大幅降低微孔体积(可降至1fL～10pL)，从而显著降低最低试剂的使用量和耗材价格。
另外，根据本发明上述实施例的基于微孔阵列芯片的数字PCR扩增装置还可以具有如下附加的技术特征：
在本发明的一些实施例中，所述电极包括片上电极、溶液电极和外接电极中的至少一种，其中，所述片上电极或所述溶液电极设置在所述底板上。
在本发明的一些实施例中，所述生化传感器为离子敏感场效应晶体管，碳纳米管，硅纳米线、石墨烯或二硫化钼晶体管传感器中的一种，或微型电化学传感器。
在本发明的一些实施例中，所述生化传感器表面设有钝化层，所述钝化层形成在所述微孔的内壁上，所述捕获探针设置在所述钝化层的表面上。
在本发明的一些实施例中，所述钝化层为金、三氧化二铝、二氧化铪、二氧化钛、五氧化二钽、二氧化硅、碳化硅的一种或多种的叠加。
在本发明的一些实施例中，每个所述生化传感器表面上设置有1-100万个所述捕获探针。
在本发明的一些实施例中，还包括：读取装置，所述读取装置与计算机电气连接，以便实现所述微孔阵列芯片PCR扩增前及PCR扩增后微孔内生化传感器电信号的读取。
在本发明的一些实施例中，所述读取装置包括：
底座，所述底座的上表面上形成有用于容纳所述微孔阵列芯片的凹槽，所述凹槽的底壁内设置有金属探针，所述金属探针适于与所述微孔阵列芯片的引脚pad形成电气连接，从而实现对微孔阵列芯片的供电及生化传感器电信号的数据读取；
电路板，所述电路板设置在所述底座的下表面上，所述电路板包括依次相连的数据采集模块、ADC芯片和处理器，其中，
所述数据采集模块与所述金属探针相连，且适于采集所述生化传感器的电信号；
所述电信号被输送至所述ADC芯片，并由所述ADC芯片进行模数转换；
经过所述模数转换的电信号被输送给所述处理器，由所述处理器进行数字信号处理并计算出扩增后DNA的浓度；
上盖，所述上盖的一侧边缘铰接在所述底座的边缘处，用于封盖所述底座。
在本发明的一些实施例中，所述读取装置包括：
插卡电路板，所述插卡电路板包括用于容纳微孔阵列芯片的容纳仓、置于容纳仓仓口的压盖、用于放大所述微孔阵列芯片上所述生化传感器信号的信号放大器芯片以及插头；
数据处理电路板，所述数据处理电路板包括插槽、ADC模数转换芯片、微处理器MCU和通信端口，所述插槽与所述插头相配，通过所述插头与所述插槽的连接实现所述插卡电路板与所述数据处理电路板的通信，所述ADC模数转换芯片分别于所述信号放大器芯片和所述微处理器MCU相连，所述微处理器MCU通过通信端口和终端设备相连。
在本发明的一些实施例中，进一步包括：
热循环器，所述热循环器包括端盖和基座，所述端盖活动安装在所述基座的顶部；所述基座包括用于容纳所述微孔阵列芯片的放置腔、紧挨所述放置腔底部设置的导热块、设置在所述导热块上端部紧挨所述放置腔位置处的温度传感器、置于所述导热块底部的加热 器、以及置于所述加热器底部的散热器；所述导热块两侧通过限位板固定在所述基座内，所述加热器通过定位板固定在所述导热块的底部，所述温度传感器和所述加热器通过控制电路与主控板相连。
根据本发明的第二方面，本发明还提出了利用前面实施例的所述数字PCR扩增装置进行扩增的方法，根据本发明的实施例，该方法包括：
(1)将DNA反应体系滴加到所述微孔阵列芯片中的每个微孔内，并用所述密封盖密封；
(2)将加样后的所述微孔阵列芯片放入读写装置中，读出所述微孔阵列芯片的初始电信号；
(3)将所述微孔阵列芯片置于热循环器上进行PCR扩增反应；
(4)将所述PCR扩增反应后的微孔阵列芯片置于读取装置内，利用所述读取装置读取所述微孔底部生化传感器的目标电信号；
(5)通过数据处理后获得DNA样本的初始浓度，并显示在终端设备上。
根据本发明的第三方面，本发明还提出了利用前面实施例的所述数字PCR扩增装置进行扩增的方法，根据本发明的实施例，该方法包括：
(1)将所述微孔阵列芯片放入读写装置中，再加入标准液到微孔后，读出所述微孔阵列芯片的初始电信号；
(2)取出所述微孔阵列芯片并去掉所述标准液后，将DNA反应体系滴加到所述微孔阵列中的每个所述微孔内，并用所述密封盖密封；
(3)将加样后的所述微孔阵列芯片置于热循环器上进行PCR扩增反应；
(4)将所述PCR扩增反应后的微孔阵列芯片清洗后，置于读取装置内，再加入所述标准液到微孔后，读出微孔阵列芯片的目标电信号；
(5)通过数据处理后获得所述DNA样本的初始浓度，并显示在终端设备上。
在本发明的一些实施例中，上述步骤(5)按照下列步骤进行：
(5-1)直方图分析：对于每一个生化传感器，用所述目标电信号减去所述初始电信号获得每个所述微孔内的PCR扩增反应前后的所述DNA电荷的差值电信号，再对所述差值电信号的数据进行所述直方图Histogram分析，从而获得所述差值电信号的分布峰：
若某个所述微孔中没有发生PCR扩增反应，则所述生化传感器的捕获探针未捕获到目 标核酸，则所述微孔内所述生化传感器在PCR扩增反应前后的所述差值电信号的值为0，未发生PCR扩增反应的微孔为阴性反应单元；
若所述微孔中发生了单个DNA样本扩增反应，则所述生化传感器的捕获探针捕获到所述目标核酸，则所述微孔内所述生化传感器在所述PCR扩增反应前后的差值电信号的值为第一电信号，发生单个所述DNA样本扩增反应的微孔为阳性反应单元；
若所述微孔内的所述生化传感器输出值的绝对值大于所述第一电信号值的绝对值，则此微孔内为多个所述DNA样本发生所述PCR扩增反应；
(5-2)所述DNA样本初始浓度的计算：由步骤(5-1)获得的所述阳性反应单元个数或所述阴性反应单元个数计算所述DNA样本的初始浓度。
在本发明的一些实施例中，上述步骤(5-2)按照下列步骤进行：
绘制标准曲线：根据各组所述DNA样本的浓度和对应的所述阳性反应单元个数N绘制标准曲线C＝f(N)；
数据计算：将待测浓度的所述DNA样本进行所述PCR扩增反应后，首先根据步骤(5-1)计算所述微孔阵列芯片上所有的所述阳性反应单元数量，然后根据预先绘制的标准曲线计算得到所述DNA样本的初始浓度。
在本发明的一些实施例中，上述步骤(5-2)按照下列步骤进行：
将所述直方图分析获得的所述阳性反应单元个数或所述阴性反应单元个数带入泊松方程计算所述DNA样本的初始浓度。
附图说明
图1是本发明实施例的微孔阵列芯片本体结构示意图；
图2中的(a)本发明实施例的芯片座式读取装置的结构示意图(关闭状态立体图)；
图2中的(b)本发明实施例的芯片座式读取装置的结构示意图(关闭状态俯视图)；
图2中的(c)本发明实施例的芯片座式读取装置的结构示意图(关闭状态主视图)；
图2中的(d)本发明实施例的芯片座式读取装置的结构示意图(打开状态侧视图)；
图2中的(e)本发明实施例的芯片座式读取装置的结构示意图(打开状态立体图)；
图2中的(f)是图2中的(c)中沿A-A沿剖视图；
图2中的(g)图2中的(f)中部分放大示意图；
图3本发明实施例的芯片座式读取装置组成框图；
图4中的(a)本发明实施例的插卡式读取装置的总体结构示意图；
图4中的(b)本发明实施例的插卡式读取装置中插卡电路板的结构示意图；
图5本发明实施例的插卡式读取装置的组成框图；
图6本发明实施例的热循环器结构示意图；
图7本发明实施例的热循环器立体示意图；
图8本发明实施例中方法(一)的半导体芯片微孔PCR示意图；
图9本发明实施例中方法(二)的半导体芯片微孔PCR示意图；
图10中的(a)本发明实施例1的PCR仪适配器示意图；
图10中的(b)为图10中的(a)的PCR仪适配器的截面示意图；
图10中的(c)为图10中的(b)的PCR仪适配器的局部放大示意图；
图11本发明之实施例1的示范数据数字二维热力图；
图12本发明实施例1的DNA探针连接前后(即表面化学前后)的电信号变化(I/V曲线)；
图13本发明实施例1的PCR扩增前后传感器信号变化的直方图；
图14本发明实施例1的阳性单元PCR前后的电信号变化(I/V曲线)图；
图15本发明实施例1的阳性单元数量与DNA浓度标准曲线图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
根据本发明的一个方面，本发明提出了基于微孔阵列芯片的数字PCR扩增装置。
下面参考附图对本发明实施例的基于微孔阵列芯片的数字PCR扩增装置进行详细描述：
如图1、图8和图9所示，该数字PCR扩增装置包括：微孔阵列芯片，所述微孔阵列芯片包括：
底板110，所述底板110上形成有微孔阵列111；密封盖120，所述密封盖120置于所 述底板110上方，用于密封所述微孔阵列111中的每个微孔112；多个生化传感器，所述微孔阵列111中每个微孔112内均设置有一个所述生化传感器，所述生化传感器表面上设置有捕获探针131，所述捕获探针适于捕获所在微孔内的目标核酸；电极，所述电极适于向所述微孔内的溶液提供电压。
由此，本发明上述实施例的数字PCR扩增装置的生化传感器通过其表面设置捕获探针来捕获扩增后的目标核酸分子，通过检测核酸分子的固有负电荷的电子检测方式，来代替荧光检测的传统方式。本申请的上述装置可以显著降低系统的复杂度、体积和价格。另外，本发明可大幅降低微孔体积(可降至1fL～10pL)，从而显著降低最低试剂的使用量和耗材价格。
根据本发明的实施例，参照图9，电极包括片上电极141、溶液电极142和外接电极143中的至少一种，其中，片上电极141或溶液电极142设置在底板110上。由此，可以通过多种形式为微孔中的溶液提供电压。
根据本发明的实施例，所述微孔阵列111中含有0.1万～1000万个微孔112，每个微孔112的容积为1fl～10pL，在微孔112内加入DNA样本及扩增液后，使用密封盖120进行压紧密封，使得不同微孔112中的液体被相互隔离，且每个微孔112内有一个的目标核酸分子，由此实现DNA样本的单分子PCR扩增。
根据本发明的实施例，每个所述生化传感器表面上设置有1个-100万个所述捕获探针。由此，捕获探针的分布密度较大，可以捕获较多的目标核酸，进而产生更大的电信号。根据本发明的实施例，所述生化传感器可以为离子敏感场效应晶体管(ISFET)，纳米线、石墨烯或二硫化钼晶体管传感器中的一种，或微型电化学传感器。具体地，生化传感器采样新型的纳米线作为沟道的场效应晶体管nanowire FET或者石墨烯或二硫化钼等二维半导体材料为沟道的FET，相比ISFET，这些纳米晶体管传感器能提供更高的灵敏度，从而提供更小的反应腔体，更高的集成度和准确性；微型电化学传感器可以大大降低扩增装置的生产成本。
根据本发明的实施例，参照图9，离子敏感场效应晶体管(ISFET)包括金属浮栅结构132、沟道133、源极134、漏极135和硅衬底136。由此，采用CMOS工艺制作ISFET，可以在微小的芯片上集成大量的生化传感器，大幅提高微孔的数量，从而提高扩增数量、检测灵敏度、准确度和动态范围。
根据本发明的实施例，参照图8和图9，生化传感器表面设有钝化层150，所述钝化层150形成在微孔112的内壁上，捕获探针131连接到钝化层的表面上。根据生化传感器的表面积大小，可以设置最少1个捕获探针，最多100万个捕获探针，比如纳米线可以涂一个到几十个捕获探针；如果是比较大的表面积，比如微孔面积2个微米，可以涂成千上万个捕获探针。
其中，捕获探针131连接到钝化层的表面上的方法可以采用表面化学的方法将捕获探针连接到钝化层的表面上，具体的：在一些实施例中，直接将巯基DNA探针直接通过巯基固载到钝化层表面；在另一些实施例中，通过在DNA上连接biotin(生物素)，由biotin再连接到钝化层上镀有avidin(亲和素)的材料上，也能形成捕获探针牢固地形成在钝化层表面上；在又一些实施例中，在DNA的一端接上胺基，再通过胺基和微孔阵列芯片上涂有醛基的单分子结合，从而固载到钝化层表面。
由此，钝化层不仅可以防止微孔中的溶液与生化传感器等其他结构接触，保证测试的准确性以及防止生化传感器中的金属材料面受溶液的腐蚀；而且，钝化层还可用于承载捕获探针。
其中，所述钝化层150为金、三氧化二铝、二氧化铪、二氧化钛、五氧化二钽、二氧化硅、碳化硅的一种或多种的叠加。由此，钝化层不会与溶液反应。
另外，本发明上述实施例的数字PCR扩增装置还包括：读取装置，所述读取装置与计算机电气连接，以便实现微孔阵列芯片PCR扩增前及PCR扩增后微孔内生化传感器电信号的读取。
其中，作为一种优选的技术方案，如图2和图3所示，所述读取装置可以为一种基于芯片座的芯片座式读取装置200，芯片座式读取装置200具体包括：底座210、电路板220和上盖230。电路板220设置在底座210的下表面上，上盖的一侧边缘铰接在底座的边缘处，用于封盖所述底座。
具体地：底座210的上表面上形成有用于容纳所述微孔阵列芯片100的凹槽211，所述凹槽211的底壁内设置有金属探针212，所述金属探针212适于与所述微孔阵列芯片的引脚pad形成电气连接，从而实现对微孔阵列芯片100的供电及生化传感器130电信号的数据读取；
电路板220，所述电路板220包括依次相连的数据采集模块、ADC芯片和处理器(未 示出)，其中，所述数据采集模块与所述金属探针相连，且适于采集所述生化传感器的电信号；所述电信号被输送至所述ADC芯片，并由所述ADC芯片进行模数转换；经过所述模数转换的电信号被输送给所述处理器，由所述处理器进行数字信号处理并计算出扩增后DNA的浓度。
根据本发明的具体实施例，上述电路板220具有的处理器可以为FPGA、DSP、或ARM。
作为另外一种技术方案，如图4中的(a)-图4中的(b)和图5所示，所述读取装置可以是基于插卡式芯片PCB电路板的插卡式读取装置300，具体包括插卡电路板310和数据处理电路板320。
具体地，插卡电路板310包括用于容纳微孔阵列芯片的容纳仓311、置于容纳仓仓口的压盖312、用于放大所述微孔阵列芯片100上所述生化传感器130信号的信号放大器芯片313以及插头314。
使用时，生化传感器130的管脚焊接在插卡电路板310上，实现对生化传感器130的电路连接，所述信号放大器芯片313将生化传感器130输出的电信号进行放大处理，提高信号噪声比后传输给数据处理电路板320的ADC模数转换芯片322；
具体地，数据处理电路板320包括插槽321、ADC模数转换芯片322、微处理器MCU323和通信端口，所述插槽321与所述插头314相配，通过所述插头314与所述插槽321的连接实现插卡电路板310与数据处理电路板320的通信，所述ADC模数转换芯片322分别于所述信号放大器芯片313和微处理器MCU323相连，所述微处理器MCU323通过通信端口和终端设备相连。具体地，通信端口可以包括图4中的(b)所示的USB端口324、串行端口325或并行端口326。终端设备可以是电脑，也可以是智能手机。
使用时，只需将插卡电路板310的插头314插入数据处理电路板320的插槽321中，即可实现插卡电路板310和数据处理电路板320的通信连接，进而通过数据处理电路板对接收到生化传感器130上的信号进行数据处理，获得并计算出扩增后DNA的浓度。
如图6和图7所示，根据本发明的实施例，本发明上述实施例的数字PCR扩增装置还可以进一步包括：热循环器400。由此可以将微孔阵列芯片100置于热循环器400上进行扩增反应。
具体地，热循环器400包括端盖410和基座，所述端盖410活动安装在所述基座的顶部；所述基座包括用于容纳微孔阵列芯片本体100的放置腔420、紧挨所述放置腔420底 部设置的导热块430、设置在所述导热块430上端部紧挨放置腔420位置处的温度传感器440、置于所述导热块430底部的加热器450、以及置于所述加热器450底部的散热器460；所述导热块430两侧通过限位板470固定在所述基座内，所述加热器450通过定位板480固定在所述导热块430的底部，所述温度传感器440和所述加热器450通过控制电路与主控板490相连。
使用时，由主控板490根据PCR扩增反应的需要设置放置腔420内的温度，温度传感器440用于监测放置腔420内的温度传输给主控板490，由主控板490根据放置腔420内的实际温度向加热器450发送加热或停止加热的命令；其中加热器450可以是半导体加热器也可以是热电阻。
根据本发明的第二方面，本发明还提出了利用前面实施例的所述数字PCR扩增装置进行扩增的方法，根据本发明的实施例，该方法(一)包括：
(1)将DNA反应体系滴加到所述微孔阵列芯片中的每个微孔内，并用所述密封盖密封；
(2)将加样后的所述微孔阵列芯片放入读写装置中，读出所述微孔阵列芯片的初始电信号；
(3)将加样后的所述微孔阵列芯片置于热循环器上进行PCR扩增反应；
(4)将PCR扩增反应后的微孔阵列芯片置于读取装置内，利用所述读取装置读取所述微孔底部生化传感器的目标电信号；
(5)通过数据处理后获得DNA样本的初始浓度，并显示在终端设备上。下面对本发明具体实施例的扩增方法(一)进行详细描述：
根据本发明的具体实施例，上述步骤(1)加样，具体包括：将目标DNA片段3根据其原有样本中的浓度情况稀释或者浓缩一定比例后，与含有引物5对及扩增酶4的缓冲液混合均匀后加入到微孔阵列芯片上的微孔112中，用密封盖120压紧密封，每个微孔112形成相互隔离的PCR反应单元，如图8所示。
步骤(3)扩增，具体包括：步骤(1)加样后的微孔阵列芯片100可以进行恒温PCR和变温PCR扩增反应，如将微孔阵列芯片100置于变温PCR仪或热循环器400上可实现变温PCR扩增反应，将微孔阵列芯片100置于恒温PCR仪或恒温台上即可实现恒温PCR扩增反应；将密封好的微孔阵列芯片100置于PCR仪或热循环器内进行PCR扩增反应； 每个微孔112中的目标DNA片段3在引物对5和扩增酶4的作用下进行链式PCR扩增反应，拷贝数持续增加，并不断地被捕获探针识别捕获，并同时释放电信号。
其中，PCR仪为平板PCR或者配合使用PCR适配器在传统PCR仪进行扩增，如96孔板。
步骤(4)信号读取，具体包括：将PCR扩增反应后的微孔阵列芯片本体置于读取装置的凹槽内，读取微孔底部生化传感器的电信号、通过数据处理后获得扩增后DNA的浓度在计算机或显示屏上显示。具体为，首先将上盖打开，将PCR扩增反应后的密封状态的微孔阵列芯片本体放入底座上的凹槽中，盖上上盖，使得密封盖压紧微孔；微孔阵列芯片本体底部生化传感器的引脚pad与底座内的探针形成电气连接，再将探针连接到电路板中，从而对微孔阵列芯片本体进行供电；然后数据采集模块采集微孔底部生化传感器的电信号，由ADC芯片进行模数转换后发生给FPGA、DSP、ARM等处理器处理进行数字信号计算处理，然后将计算获得DNA样本的初始浓度发送给显示屏显示。
根据本发明的具体实施例，上述步骤(5)按照下列步骤进行：
(5-1)直方图分析：对于每一个生化传感器，用所述目标电信号减去所述初始电信号获得每个所述微孔内的PCR扩增反应前后的所述DNA电荷的差值电信号，再对所述差值电信号的数据进行直方图Histogram分析，从而获得差值电信号的分布峰：若某个微孔中没有发生PCR扩增反应，则所述生化传感器的捕获探针未捕获到目标核酸，则所述微孔内所述生化传感器在PCR扩增反应前后的差值电信号的值为0，未发生PCR扩增反应的微孔为阴性反应单元；若微孔中发生了单个DNA样本扩增反应，则所述生化传感器的捕获探针捕获到目标核酸，则所述微孔内所述生化传感器在PCR扩增前后的差值电信号的值为第一电信号值，发生单个DNA样本PCR扩增反应的微孔为阳性反应单元；若微孔内的生化传感器输出值的绝对值大于所述第一电信号值的绝对值，则此微孔内为多个DNA样本发生PCR扩增反应；
(5-2)DNA样本初始浓度的计算：由步骤(5-1)获得的阳性反应单元个数或阴性反应单元个数计算DNA样本的初始浓度。
根据本发明的具体实施例，上述步骤(5-2)按照下列步骤进行：
绘制标准曲线：根据各组DNA样本的浓度和对应的阳性反应单元个数N绘制标准曲线C＝f(N)；
数据计算：将待测浓度的DNA样本进行PCR扩增反应后，首先根据步骤(5-1)计算微孔阵列芯片上所有的阳性反应单元数量，然后根据预先绘制的标准曲线计算得到DNA样本的初始浓度。
根据本发明的具体实施例，上述步骤(5-2)还可以按照下列步骤进行：
将直方图分析获得的阳性反应单元个数或阴性反应单元个数带入泊松方程计算DNA样本的初始浓度。
根据本发明的第三方面，本发明还提出了利用前面实施例的所述数字PCR扩增装置进行扩增的方法，根据本发明的实施例，参照图9，该方法(二)包括：
(1)将所述微孔阵列芯片放入读写装置中，再加入标准液6到微孔后，读出微孔阵列芯片的初始电信号；
(2)取出所述微孔阵列芯片并去掉标准液6后，将DNA反应体系滴加到所述微孔阵列中的每个微孔内，并用所述密封盖密封；
(3)将加样后的所述微孔阵列芯片置于热循环器上进行PCR扩增反应，得到PCR扩增反应后DNA 7，参照图9；
(4)将所述PCR扩增反应后的微孔阵列芯片清洗后，置于读取装置内，再加入标准液6到微孔后，读出微孔阵列芯片的目标电信号；
(5)通过数据处理后获得所述DNA样本的初始浓度，并显示在终端设备上。
其中，该方法(二)中的步骤(5)与前面所述方法(一)中的步骤(5)的操作分析步骤一致，在此不再过多的赘述。
实施例
为了更好的说明本发明提供的扩增装置的使用方法，现以CMOS ISFET作为生化传感器的微孔阵列芯片本体作为实施例进行具体说明，具体指标如表1：
表1 微孔阵列芯片本体的指标
传感器种类 ISFET
微孔及传感器数量 ～26万个
微孔尺寸(长，宽，深度) 2微米，2微米，1.5微米
传感器表面感应材料种类 Ta 2O 5/Au
传感器表面感应材料厚度 20nm
检测PCR的信号类型 氢离子浓度，或扩增后DNA的电荷
1.捕获探针连接芯片
捕获探针(也可称为DNA探针)连接微孔阵列芯片的操作按如下步骤进行：a、将含有捕获探针的连接液(见表2)混匀后取2.5uL滴加到芯片上；b、将微孔阵列芯片放入真空装置内抽真空(0.1M Pa)并保持1分钟后取出；c、在室温保湿1小时后用去离子水洗掉未连接的捕获探针。
表2：捕获探针连接液配制
NaCl 1M
Tris-HCl 50mM
巯基PEG4 1mM
TCEP 1mM
巯基DNA探针 10uM
pH 7.5
2.上样
如图9所示，将6个不同已知浓度的目标DNA溶液(0.01PM，0.1PM,1PM,10PM,100PM,1NM)分别与含有DNA聚合酶、dNTPs和引物的PCR扩增液混合均匀。溶液混合量及比例如表3所示，10微升体系可用于四张芯片。按如下步骤上样：a、将混合后的溶液取2.5uL滴加到微孔阵列芯片上(每个特定浓度溶液对应一个芯片)，使每个微孔内均含有混合溶液；b、将芯片放入真空装置内抽真空(0.1M Pa)并保持1分钟后取出；c、将微孔阵列芯片100(也可简称为芯片)放入图10所示的PCR锥形适配器里；d、用密封盖密封半导体芯片表 面并合上PCR适配器上盖1030，将密封压片与微孔阵列芯片100微孔上表面压紧密封，使得微孔阵列芯片100本体的每个微孔均是独立的反应空间。
其中，参照图10中的(a)、(b)和(c)，PCR锥形适配器可单独或者多个一起放置在PCR仪器的加热槽内进行加热，对放置在微孔阵列芯片100上的液体进行PCR扩增；PCR锥形适配器的导热性能佳，可快速将PCR仪器的加热槽上的热量传导至芯片上；芯片可分离的设置在PCR锥形适配器上，使芯片PCR适配器完成不同芯片的PCR扩增。PCR锥形适配器包括：基座1010、上盖1030、堵头1040、卡扣1060和弹簧1070，具体的：包括与基座1010扣合连接的上盖1030，上盖1030上设置有压合在芯片上的堵头1040，上盖1030的外侧设置有扣合在基座上的卡扣1060。卡扣1060的中部枢轴连接在上盖1030上，卡扣1060的上端与弹簧1070的一端固定相连，弹簧1070的另一端固定在上盖1030上。人工拨动卡扣1060可将上盖1030扣合在基座1010上，当上盖1030扣合在基座1010上后，堵头1040将芯片100压合在芯片放置槽内，使芯片100位置固定。
表3：PCR溶液配制
DNA Template 0.01pM到1nM
PCR MIX 5ul
Primer 适量
去离子H 2O 至10ul
3.PCR扩增反应
将一个或多个含有芯片本体的PCR适配器放到普通PCR仪上的96孔板的孔洞内，适配器底座为金属或导热材料，对芯片本体进行导热，进行PCR扩增，扩增条件如表4所示：
表4：PCR扩增条件
Figure PCTCN2020102374-appb-000001
Figure PCTCN2020102374-appb-000002
4.信号读取
PCR扩增完成后将带有密封压片的芯片(保持密封状态)从PCR适配器中取出后有2种方法对PCR扩增产生的电信号进行读取：方法a、将芯片取出后放置在如图2和图3所示的读取装置的socket内，直接通过读取装置(通过片上电极给芯片的微孔溶液提供基准电压)的电路连接芯片管脚，将所有微孔内传感器的电信号经数据处理读出到计算机或到显示屏；为防止方法a中的PCR扩增结束后PCR溶液发生变化会导致可能发生两种信号，一种是DNA本身的电荷信号，另外一个是PCR溶液本身发生变化引起的另外一个假信号，从而导致测得的结果不准确，进一步地，可以采用方法b、将芯片取出后用pH8.0缓冲液或去离子水或纯净水进行冲洗，将原先的溶液清洗掉，然后再将加有pH8.0缓冲液或去离子水或纯净水的芯片放置在如图2和图3所示的读取装置的socket内，这样使得PCR溶液测之前和测之后没有任何变化，最后通过读取装置(通过片上电极或外接电极给芯片的微孔溶液提供基准电压)的电路连接芯片管脚，将所有微孔内传感器的电信号经数据处理读出到计算机或到显示屏。
对芯片本体进行数据驱动过程中，每一个时间点，都产生26万个数据，作为一帧，分别对应26个生化传感器。数据按512行×512列，形成一个二维热力图，如图11所示，每一个像素的颜色对应相应传感器的输出信号的大小，即其对应的微孔内的pH值/DNA电荷。多个时间点可产生多帧二维热力图。
具体过程如下：
(1)DNA探针连接前后：首先在DNA探针连接前后分别获取芯片的数帧二维热力图数据，对微孔内连接DNA探针前后的多帧电信号进行比较。如图12所示微孔的电信号变化明显，即为微孔内连有DNA探针，其中，图12中的上曲线A1表示DNA探针连接之前，下曲线A2表示DNA探针连接之后。
(2)PCR扩增前后：对于各个初始DNA浓度(C1＝0.01pM到1nM)的6个样本，对应的6颗芯片本体，每颗芯片本体都获得PCR扩增前后的数帧二维热力图数据。首先，对微孔内PCR扩增前后的多帧电信号进行比较。其次，对每个芯片本体求得所有微孔的电信号变化并进行直方图Histogram分析。如图13所示，Histogram分析获得多个分布峰：没有DNA样本发生PCR扩增的微孔，其电信号没有发生变化，传感器输出为0，则相应微孔为 阴性反应单元，即阴性传感器；再次，有一个或多个DNA样本发生PCR反应的微孔中电信号发生变化，则相应微孔为阳性反应单元，即阳性传感器，如图14所示微孔的电信号变化明显，即为微孔内有DNA模板进行了PCR扩增(图14中上曲线B1为PCR扩增之后，下曲线B2为PCR扩增之前)。如果溶液稀释比例合适，多个DNA样本发生PCR反应干扰的单元数量很少。
将各个初始DNA浓度(C1＝0.01pM到1nM)和对应的阳性传感器的数量N绘制如图15所示的标准曲线C＝f(N)。
再根据待测样本在芯片本体上的信号所计算出的阳性传感器数量，带入标准曲线方程，计算得到DNA样本的初始拷贝浓度(C2)。通过对比泊松公式计算的结果，所测DNA拷贝浓度C2与实际拷贝浓度C1较为接近，从而验证所获得的标准曲线与泊松分布公式一致。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (15)

  1. 一种基于微孔阵列芯片的数字PCR扩增装置，其特征在于，包括：微孔阵列芯片，所述微孔阵列芯片包括：
    底板，所述底板上形成有微孔阵列；
    密封盖，所述密封盖置于所述底板上方，用于密封所述微孔阵列中的每个微孔；
    多个生化传感器，所述微孔阵列中每个微孔内均设置有一个所述生化传感器，所述生化传感器表面上设置有捕获探针，所述捕获探针适于捕获所在微孔内的目标核酸；
    电极，所述电极适于向所述微孔内的溶液提供电压。
  2. 根据权利要求1所述的基于微孔阵列芯片的数字PCR扩增装置，其特征在于，所述电极包括片上电极、溶液电极和外接电极中的至少一种，其中，所述片上电极或所述溶液电极设置在所述底板上。
  3. 根据权利要求1所述的基于微孔阵列芯片的数字PCR扩增装置，其特征在于，所述生化传感器为离子敏感场效应晶体管，碳纳米管，硅纳米线、石墨烯或二硫化钼晶体管传感器中的一种，或微型电化学传感器。
  4. 根据权利要求1所述的基于微孔阵列芯片的数字PCR扩增装置，其特征在于，所述生化传感器表面上设有钝化层，所述钝化层形成在所述微孔的内壁上，所述捕获探针设置在所述钝化层的表面上。
  5. 根据权利要求1或3所述的基于微孔阵列芯片的数字PCR扩增装置，其特征在于，所述钝化层为金、三氧化二铝、二氧化铪、二氧化钛、五氧化二钽、二氧化硅、碳化硅中的一种或多种的叠加。
  6. 根据权利要求1或3所述的基于微孔阵列芯片的数字PCR扩增装置，其特征在于，每个所述生化传感器表面上设置有1-100万个所述捕获探针。
  7. 根据权利要求1所述的基于微孔阵列芯片的数字PCR扩增装置，其特征在于，还包括：
    读取装置，所述读取装置与计算机电气连接，以便实现所述微孔阵列芯片PCR扩增前及PCR扩增后所述微孔内的所述生化传感器电信号的读取。
  8. 根据权利要求7所述的基于微孔阵列芯片的数字PCR扩增装置，其特征在于，所述读取装置包括：
    底座，所述底座的上表面上形成有用于容纳所述微孔阵列芯片的凹槽，所述凹槽的底壁内设置有金属探针，所述金属探针适于与所述微孔阵列芯片的引脚pad形成电气连接，从而实现对微孔阵列芯片的供电及生化传感器电信号的数据读取；
    电路板，所述电路板设置在所述底座的下表面上，所述电路板包括依次相连的数据采集模块、ADC芯片和处理器，其中，
    所述数据采集模块与所述金属探针相连，且适于采集所述生化传感器的电信号；
    所述电信号被输送至所述ADC芯片，并由所述ADC芯片进行模数转换；
    经过所述模数转换的电信号被输送给所述处理器，由所述处理器进行数字信号处理并计算出扩增后DNA的浓度；
    上盖，所述上盖的一侧边缘铰接在所述底座的边缘处，用于封盖所述底座。
  9. 根据权利要求7所述的基于微孔阵列芯片的数字PCR扩增装置，其特征在于，所述读取装置包括：
    插卡电路板，所述插卡电路板包括用于容纳微孔阵列芯片的容纳仓、置于容纳仓仓口的压盖、用于放大所述微孔阵列芯片上所述生化传感器信号的信号放大器芯片以及插头；
    数据处理电路板，所述数据处理电路板包括插槽、ADC模数转换芯片、微处理器MCU和通信端口，所述插槽与所述插头相配，通过所述插头与所述插槽的连接实现所述插卡电路板与所述数据处理电路板的通信，所述ADC模数转换芯片分别与所述信号放大器芯片和所述微处理器MCU相连，所述微处理器MCU通过通信端口和终端设备相连。
  10. 根据权利要求1所述的基于微孔阵列芯片的数字PCR扩增装置，其特征在于，进一步包括：
    热循环器，所述热循环器包括端盖和基座，所述端盖活动安装在所述基座的顶部；所述基座包括用于容纳所述微孔阵列芯片的放置腔、紧挨所述放置腔底部设置的导热块、设置在所述导热块上端部紧挨所述放置腔位置处的温度传感器、置于所述导热块底部的加热器、以及置于所述加热器底部的散热器；所述导热块两侧通过限位板固定在所述基座内，所述加热器通过定位板固定在所述导热块的底部，所述温度传感器和所述加热器通过控制电路与主控板相连。
  11. 一种利用权利要求1-10任一项所述数字PCR扩增装置进行扩增的方法，其特征在于，包括：
    (1)将DNA反应体系滴加到所述微孔阵列芯片中的每个微孔内，并用所述密封盖密封；
    (2)将加样后的所述微孔阵列芯片放入读写装置中，读出所述微孔阵列芯片的初始电信号；
    (3)将加样后的所述微孔阵列芯片置于热循环器上进行PCR扩增反应；
    (4)将所述PCR扩增反应后的微孔阵列芯片置于读取装置内，利用所述读取装置读取所述微孔底部生化传感器的目标电信号；
    (5)通过数据处理后获得所述DNA样本的初始浓度，并显示在终端设备上。
  12. 一种利用权利要求1-10任一项所述数字PCR扩增装置进行扩增的方法，其特征在于，包括：
    (1)将所述微孔阵列芯片放入读写装置中，再加入标准液到微孔后，读出所述微孔阵列芯片的初始电信号；
    (2)取出所述微孔阵列芯片并去掉所述标准液后，将DNA反应体系滴加到所述微孔阵列中的每个所述微孔内，并用所述密封盖密封；
    (3)将加样后的所述微孔阵列芯片置于热循环器上进行PCR扩增反应；
    (4)将所述PCR扩增反应后的微孔阵列芯片清洗后，置于读取装置内，再加入所述标准液到微孔后，读出所述微孔阵列芯片的目标电信号；
    (5)通过数据处理后获得所述DNA样本的初始浓度，并显示在终端设备上。
  13. 根据权利要求11或12所述的方法，其特征在于，步骤(5)按照下列步骤进行：
    (5-1)直方图分析：对于每一个传感器，用所述目标电信号减去所述初始电信号获得每个所述微孔内的PCR扩增反应前后的所述DNA电荷的差值电信号，再对所述差值电信号的数据进行所述直方图Histogram分析，从而获得所述差值电信号的分布峰：
    若某个所述微孔中没有发生PCR扩增反应，则所述生化传感器的捕获探针未捕获到目标核酸，则所述微孔内所述生化传感器在所述PCR扩增反应前后的所述差值电信号的值为0，未发生所述PCR扩增反应的微孔为阴性反应单元；
    若所述微孔中发生了单个DNA样本扩增反应，则所述生化传感器的捕获探针捕获到所述目标核酸，则所述微孔内所述生化传感器在所述PCR扩增反应前后的所述差值电信号的值为第一电信号值，发生单个所述DNA样本扩增反应的微孔为阳性反应单元；
    若所述微孔内的所述生化传感器输出值的绝对值大于所述第一电信号值的绝对值，则此微孔内为多个所述DNA样本发生所述PCR扩增反应；
    (5-2)所述DNA样本初始浓度的计算：由步骤(5-1)获得的所述阳性反应单元个数或所述阴性反应单元个数计算所述DNA样本的初始浓度。
  14. 根据权利要求13所述的方法，其特征在于，步骤(5-2)按照下列步骤进行：
    绘制标准曲线：根据各组所述DNA样本的浓度和对应的所述阳性反应单元个数N绘制标准曲线C＝f(N)；
    数据计算：将待测浓度的所述DNA样本进行所述PCR扩增反应后，首先根据步骤(5-1)计算所述微孔阵列芯片上所有的所述阳性反应单元数量，然后根据预先绘制的标准曲线计算得到所述DNA样本的初始浓度。
  15. 根据权利要求13所述的方法，其特征在于，步骤(5-2)按照下列步骤进行：
    将所述直方图分析获得的所述阳性反应单元个数或所述阴性反应单元个数带入泊松方程计算所述DNA样本的初始浓度。
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